E. coli MG1655 Top-Down Proteomics Summary

David Butcher

Presentation generated September 05, 2019

E. coli MG1655

Image courtesy of NIH NIAID

Image courtesy of NIH NIAID

Jensen. J. Bacteriol. **1993**, *175(11)*, 3401-3407

Jensen. J. Bacteriol. 1993, 175(11), 3401-3407

E. coli MG1655 Whole Cell Lysate

Cell Culture

  1. Streak frozen glycerol stock on appropriate agar medium and incubate 12 - 16 hours at 37 °C.
  2. Use single colonies to inoculate 2 culture tubes containing 5 mL of liquid medium. Incubate at 37 °C for ~16 hours (rich medium) or ~24 hours (minimal medium).
  3. Use 50 μL of saturated culture (1/300th volume) to inoculate 50 mL BioReaction tubes containing 15 mL of liquid medium. Incubate at 37 °C with 250 RPM shaking until OD600 = 1.0 (3 - 4 hours for rich medium, 10 - 12 hours for minimal medium).
  4. Centrifuge tubes at 4,000G for 15 mins, decant excess culture medium (into 25% bleach solution) and freeze cell pellets at -80 °C.

Cell Lysis

  1. Begin cooling the centrifuge to 4 °C.
  2. Thaw cell pellets on ice for 15 mins, then resuspend each pellet in pre-chilled lysis buffer. Use 1 mL of lysis buffer per ~5 mL of suspension culture.
  3. Pipette to mix until a marked increase in sample viscosity (due to released genomic DNA) is observed.
  4. Incubate lysate on ice for 20 mins. Swirl to mix every 5 min.
  5. Add 1 µL of 1 M MgCl2 per 5 mL of initial culture volume to the lysate (final concentration of 1 mM).
  6. Add 0.5 µL (125 U) of benzonase nuclease per 5 mL of initial culture volume to the lysate.
  7. Incubate lysate at 37 °C for 20 min. Invert to mix several times every 5 mins.
  8. Chill lysates on ice for 5 mins.
  9. Pellet remaining debris by centrifugation at 12,000G for 20 mins at 4 °C.

GELFrEE Fractionation

GELFrEE 8100 Fractionation System

GELFrEE 8100 Fractionation System

Figure courtesy of Lissa Anderson

Figure courtesy of Lissa Anderson

10% GELFrEE Fractionation of *E. coli* MG1655 WCL LB-B-20190515

10% GELFrEE Fractionation of E. coli MG1655 WCL LB-B-20190515

PEPPI Fractionation

PEPPI Fractions SDS-PAGE

1 mM DTT, 2 mM IAA PEPPI

1 mM DTT, 2 mM IAA PEPPI

5 mM DTT, 10 mM IAA PEPPI

5 mM DTT, 10 mM IAA PEPPI

1 mM DTT, 2 mM IAA PEPPI fractions SDS-PAGE

1 mM DTT, 2 mM IAA PEPPI fractions SDS-PAGE

PEPPI fractions exhibit less overlap due to better spatial separation in the initial PEPPI gel.

PEPPI Gels

Gels used for PEPPI are available in a 2D-well format!

Large lane loading volume: 400 μL

Top-Down Summary

Table 1: Overview of LC-MS/MS Runs by Fractionation Method

All protein and proteoform ID numbers were taken from TDreport files viewed in TDViewer 2.0.0. FDR cutoff was set to 1% and “characterized only” box was unchecked.

Protein IDs for unfrac. WCL, LB medium (unfrac01)

Table 2: Protein IDs for unfrac. WCL, LB medium from 201907_EcoliMG1655WCL_LB-B-20190320_9runs.tdReport

Proteoform IDs for unfrac. WCL, LB medium (unfrac01)

Table 3: Proteoform IDs for unfrac. WCL, LB medium from 201907_EcoliMG1655WCL_LB-B-20190320_9runs.tdReport

Mass Distribution Histograms (unfrac01)

Protein IDs for GELFrEE fractions, LB medium (gf02)

Table X: Protein IDs for GELFrEE fractions from 20190612_GELFrEE_F01-08.tdReport

Proteoform IDs for GELFrEE fractions, LB medium (gf02)

Table X: Proteoform IDs for GELFrEE fractions from 20190612_GELFrEE_F01-08.tdReport

Mass Distribution Histograms (gf02)

Protein IDs for GELFrEE fractions, LB medium, single trial TDsearch (gf03)

Table X: Protein IDs for GELFrEE fractions from 20190612_GELFrEE_F01-08_1run.tdReport

Proteoform IDs for GELFrEE fractions, LB medium, single trial TDsearch (gf03)

Table X: Proteoform IDs for GELFrEE fractions from 20190612_GELFrEE_F01-08_1run.tdReport

Mass Distribution Histograms (gf03)

Waffle Plot (gf03)

Protein IDs for PEPPI fractions, LB medium, TDsearch w/ carboxamidomethylation (peppi02b)

Table X: Protein IDs for PEPPI fractions from 20190626-28_PEPPI_F01-F06_1mMDTT_2mMIAA_CAMsearch.tdReport

Proteoform IDs for PEPPI fractions, LB medium, TDsearch w/ carboxamidomethylation (peppi02b)

Table X: Proteoform IDs for PEPPI fractions from 20190626-28_PEPPI_F01-F06_1mMDTT_2mMIAA_CAMsearch.tdReport

Mass Distribution Histograms (peppi02b)

Waffle Plot (peppi02b)

Protein/proteoform IDs for LB and M9 media

Figure 3: Venn diagrams for unfractionated WCL runs. (Left) Protein IDs by medium type. (Right) Proteoform IDs by medium type.

UpSet plots, all LC-MS/MS methods

Figure 4: Protein identification numbers determined for unfractionated, GELFrEE-fractionated, and PEPPI-fractionated samples.

UpSet plots, all LC-MS/MS methods

Figure 5: Proteoform identification numbers determined for unfractionated, GELFrEE-fractionated, and PEPPI-fractionated samples.

UpSet plots, GELFrEE Fractions (gf02)

Figure 6: Protein identifications determined for GELFrEE fractions (gf02).

UpSet plots, GELFrEE Fractions (gf02)

Figure 7: Proteoform identifications determined for GELFrEE fractions (gf02).

UpSet plots, PEPPI fractions (peppi02b)

Figure 8: Protein identifications determined for PEPPI fractions (peppi02b).

UpSet plots, PEPPI fractions (peppi02b)

Figure 9: Proteoform identifications determined for GELFrEE fractions (peppi02b).

Effect of multiple LC-MS/MS runs on protein/proteoform IDs

Session Info

Software and packages used to generate this report:

## R version 3.6.1 (2019-07-05)
## Platform: x86_64-w64-mingw32/x64 (64-bit)
## Running under: Windows 10 x64 (build 17763)
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## [3] LC_MONETARY=English_United States.1252
## [4] LC_NUMERIC=C                          
## [5] LC_TIME=English_United States.1252    
## 
## attached base packages:
## [1] stats     graphics  grDevices utils     datasets  methods   base     
## 
## other attached packages:
##  [1] readxl_1.3.1          DT_0.8                Vennerable_3.1.0.9000
##  [4] UpSetR_1.4.0          magrittr_1.5          forcats_0.4.0        
##  [7] stringr_1.4.0         dplyr_0.8.3           purrr_0.3.2          
## [10] readr_1.3.1           tidyr_0.8.3           tibble_2.1.3         
## [13] ggplot2_3.2.1         tidyverse_1.2.1       kableExtra_1.1.0     
## 
## loaded via a namespace (and not attached):
##  [1] httr_1.4.1          jsonlite_1.6        viridisLite_0.3.0  
##  [4] modelr_0.1.5        shiny_1.3.2         assertthat_0.2.1   
##  [7] highr_0.8           stats4_3.6.1        RBGL_1.60.0        
## [10] cellranger_1.1.0    yaml_2.2.0          pillar_1.4.2       
## [13] backports_1.1.4     lattice_0.20-38     glue_1.3.1         
## [16] digest_0.6.20       RColorBrewer_1.1-2  promises_1.0.1     
## [19] rvest_0.3.4         colorspace_1.4-1    htmltools_0.3.6    
## [22] httpuv_1.5.1        plyr_1.8.4          pkgconfig_2.0.2    
## [25] broom_0.5.2         haven_2.1.1         xtable_1.8-4       
## [28] scales_1.0.0        webshot_0.5.1       later_0.8.0        
## [31] generics_0.0.2      withr_2.1.2         BiocGenerics_0.30.0
## [34] lazyeval_0.2.2      cli_1.1.0           crayon_1.3.4       
## [37] mime_0.7            evaluate_0.14       nlme_3.1-140       
## [40] xml2_1.2.1          graph_1.62.0        tools_3.6.1        
## [43] hms_0.5.1           munsell_0.5.0       compiler_3.6.1     
## [46] rlang_0.4.0         grid_3.6.1          rstudioapi_0.10    
## [49] htmlwidgets_1.3     crosstalk_1.0.0     labeling_0.3       
## [52] rmarkdown_1.15      gtable_0.3.0        reshape2_1.4.3     
## [55] R6_2.4.0            gridExtra_2.3       lubridate_1.7.4    
## [58] knitr_1.24          zeallot_0.1.0       stringi_1.4.3      
## [61] parallel_3.6.1      Rcpp_1.0.2          vctrs_0.2.0        
## [64] png_0.1-7           tidyselect_0.2.5    xfun_0.9